Fenofibrate subcellular distribution as a rationale for the intracranial delivery through biodegradable carrier

Abstract

Fenofibrate, a well-known normolipidemic drug, has been shown to exert strong anticancer effects against tumors of neuroectodermal origin including glioblastoma. Although some pharmacokinetic studies were performed in the past, data are still needed about the detailed subcellular and tissue distribution of fenofibrate (FF) and its active metabolite, fenofibric acid (FA), especially in respect to the treatment of intracranial tumors. We used high performance liquid chromatography (HPLC) to elucidate the intracellular, tissue and body fluid distribution of FF and FA after oral administration of the drug to mice bearing intracranial glioblastoma. Following the treatment, FF was quickly cleaved to FA by blood esterases and FA was detected in the blood, urine, liver, kidney, spleen and lungs. We have also detected small amounts of FA in the brains of two out of six mice, but not in the brain tumor tissue. The lack of FF and FA in the intracranial tumors prompted us to develop a new method for intracranial delivery of FF. We have prepared and tested in vitro biodegradable poly-lactic-co-glycolic acid (PLGA) polymer wafers containing FF, which could ultimately be inserted into the brain cavity following resection of the brain tumor. HPLC-based analysis demonstrated a slow and constant diffusion of FF from the wafer, and the released FF abolished clonogenic growth of glioblastoma cells. On the intracellular level, FF and FA were both present in the cytosolic fraction. Surprisingly, we also detected FF, but not FA in the cell membrane fraction. Electron paramagnetic resonance spectroscopy applied to spin-labeled phospholipid model-membranes revealed broadening of lipid phase transitions and decrease of membrane polarity induced by fenofibrate. Our results indicate that the membrane-bound FF could contribute to its exceptional anticancer potential in comparison to other lipid-lowering drugs, and advocate for intracranial delivery of FF in the combined pharmacotherapy against glioblastoma.

Fig. 1

Chemical structures of fenofibrate (FF), fenofibric acid (FA), gemfibrozil and Wy-14,643 (pirinixic acid).

Fig. 2

Detection of fenofibrate (FF) and fenofibric acid (FA) by high performance liquid chromatography [HPLC; Agilent Technologies 1100 with on line degasser, binary pump, auto-sampler, 3 μm, 4.6 × 150 mm column YMCBase 3 μm, 4.6 × 150 mm column (octyl silane C8 chemically bonded to totally porous silica particles), thermostat, and diode array detector (DAD)]. The sample separation parameters: solvent a: 50 mM acetic acid in water, solvent b: acetonitrile, isocratic 60%; flow rate 1ml/min; temp 20°C; injection fraction 5 μl; detection: DAD at 285 nm. (Panel a): HPLC chromatogram of FF and FA standards; (panel b): FF 50 μM solution in human blood just after mixing (time 0); (panel c): the same solution as in B after 4 hours incubation at 37°C; (panel d): HPLC analysis of urine from the patient who takes fenofibrate regularly (200 mg of micronized fenofibrate daily). FA and FF retention times are 3.904 ± 0.004 min and 10.436 ± 0.084 min, respectively. Insets: UV-Vis absorption spectra for FF and FA showing absorption maxima at 285 nm.

Fig. 3

(Panel a): Schematic description of the preparation of poly-lactic-co-glycolic acid (PLGA) wafers containing fenofibrate. (Panel b): SEM image of highly porous PLGA film cast using breath figure methodology. SEM images were prepared as described in (33, 34).

Fig. 4

Fenofibrate (FF) and fenofibric acid (FA) concentrations in whole cell lysates (a) and cell culture media (b) from the monolayer culture of human glioblastoma cell line, LN-229 were treated with a single dose of fenofibrate (50 μM) FF and FA were determined at the indicated time points using as described in the materials and methods section. Data represent average values from 2 experiments in triplicate (n=6) ± S.D. (S.D. values for some points are too small to be visible).

Fig. 5

Fenofibrate (FF) and its metabolite, fenofibric acid (FA), in cell membrane and cytosolic fractions isolated from FF treated human glioblastoma cell line, LN-229. (Panel a): quantitative analysis of FF and FA in membrane (mem) and cytosolic (cyto) fractions isolated from LN-229 cells exposed to 50 μM fenofibrate (FF50) for 24 hours. Concentrations of FF and FA were calculated from the corresponding calibration curves, and are expressed in nmols of FF and FA per 1 × 10⁶ cells. Data represent average values from three separate measurements with standard deviation. (Panel b): Western blot analysis demonstrating purity of cytosolic (Cyto) and membrane (Mem) fractions in which N-cadherin and GAPDH were use as membrane and cytosolic markers, respectively.

Fig. 6

HPLC-based detection of fenofibrate and its metabolite, fenofibric acid, in cell membrane (panel a) and cystolic fraction (panel b) isolated from fenofibrate treated human glioblastoma cell line, LN-229. Under these chromatographic conditions (see legend to Fig. 2), fenofibrate was eluted at 10.4 minutes and fenofibric acid at 3.9 min. Insets in (a) and (b) represent the unique UV-Vis absorbance spectra of the obtained peaks corresponding to fenofibrate and fenofibric acid, respectively.

Fig. 7

Normalized amplitude of the central peak of the EPR spectra of 5-SASL (a) and 16-SASL (b) plotted as a function of temperature (cooling experiments) in DMPC bilayer. Experiments were performed in the absence and presence of FF (2.5 mol% and 5 mol%). T_M and dT_1/2 were obtained by fitting sigmoidal curves using Origin software.

Fig. 8

Hydrophobicity profiles (2A_z) across the DMPC membrane. Profiles were obtained for the membranes without additions and after addition of 2.5 mol% or 5 mol% of FF. Upward changes (lower values of the 2A_z parameter) indicate increase in hydrophobicity. Approximate location of the nitroxide moieties of spin labels are indicated by arrows. The numbers with stars for n-SASLs indicate that these SASLs are intercalated in the right half of the bilayer, but the nitroxide attached to C16 may pass through the center of the bilayer and stay in the other leaflet of the membrane. For more details see (38).

Fig. 9

Evaluation of PLGA wafer containing 1 mg of fenofibrate (FF). (Panel a): HPLC-based measurement of FF release to the culture media, after submerging the PLGA wafer containing 1 mg of FF. The analysis was performed in the absence of cells. (Panel b): Clonogenic growth of LN-229 cells in the presence of PLGA wafer containing 1 mg of FF (PLGA/FF); empty wafer (PLGA); and FF in DMSO applied as a single 50 μM dose (FF 50 μM). (Panel c): FF and FA concentrations in LN-229 cells cultured in the presence of PLGA/FF wafer for 12 days. (Panel d): Clonogenic assay in LN-229 cells treated with different PPARα agonists and metformin: DMSO (vehicle), FF - fenofibrate 50 μM; Met - metformin 50 μM; Gem - gemfibrozil 50 μM; Wy - Wy14,643 50 μM. (Panel e): Clonogenic assay in LN-229 treated with FF (25 μM), PPARα inhibitor MK-886 (10 μM) or both compounds together. Cells were treated with the indicated compounds for 12 days; medium with the drugs was changed every second day.